Datasheet Extract

Anti-Collagen antibodies have been used for indirect trapping ELISA for quantitation of antigen in serum using a standard curve, for Immunoprecipitation and for native (non-denaturing, non-dissociating) PAGE and Western blotting for highly sensitive qualitative analysis.Recommended Dilutions:ELISA: 1/5,000-1/50,000. Western blot: 1/1,000-1/10,000. Immunoprecipitation: 1/100. Immunohistochemistry: 1/50-1/200.QC: This product was assayed by Immunoblot and found to be reactive against Collagen IV at a dilution of 1/5,000-1/10,000. This product was also assayed against 1.0 µg of Collagen IV in a standard sandwich ELISA using Peroxidase conjugated Affinity Purified anti-Rabbit IgG [H&L] (Goat) (Cat.-No R1364HRP) and (ABTS (2,2'-azino-bis-[3-ethylbenthiazoline-6-sulfonic acid]) as a substrate for 30 minutes at room temperature. A working dilution of 1/4,000-1/8,000 of the stock concentration showed best results.

Background

Collagens are highly conserved throughout evolution and are characterized by an uninterrupted "Glycine-X-Y" triplet repeat that is a necessary part of the triple helical structure. For these reasons it is often extremely difficult to generate antibodies with specificities to collagens. The development of type specific antibodies is dependent on NON-DENATURED three-dimensional epitopes. Collagens were extensively purified for immunization from human and bovine placenta and cartilage by limited pepsin digestion and selective salt precipitation. This preparation results in a native conformation of the protein. Antibodies are isolated from rabbit antiserum and are extensively cross-adsorbed by immunoaffinity purification to produce 'type' specific antibodies. Greatly diminished reactivity and selectivity of these antibodies will result if denaturing and reducing conditions of SDS-PAGE and immunoblotting are used.

Store undiluted at 2-8°C for one month or (in aliquots) at -20°C for longer.For extended storage, mix with an equal volume of glycerol.Avoid repeated freezing and thawing.Shelf life: one year from despatch.

Format

Purification:

Immunoaffinity Chromatography

Buffer System:

0.125M Sodium Borate, 0.075M Sodium Chloride, 0.005M EDTA, pH 8.0

Preservatives:

0.01% (w/v) Sodium Azide

Stabilizers:

None

State:

Liquid (sterile filtered) purified Ig fractionAff - Purified

Specificity

Specificity:

This product has been prepared by Immunoaffinity Chromatography using immobilized antigens followed by extensive cross-adsorption against other collagens, human serum proteins and non-collagen extracellular matrix proteins to remove any unwanted specificities. Typically less than 1% cross reactivity against other types of collagens was detected by ELISA against purified standards.Some class specific anti-collagens may be specific for three-dimensional epitopes which may result in diminished reactivity with denatured collagen or formalin-fixed, paraffin embedded tissues. This antibody reacts with most mammalian Type IV collagens and has negligible cross-reactivity with Type I, II, III, V or VI collagens. Non-specific cross reaction of anti-collagen antibodies with other human serum proteins or non-collagen extracellular matrix proteins is negligible.